Bone is an active tissue, continually undergoing turnover, where there are interactive cycles of bone formation and resorption. Bone resorption is generally rapid and is mediated by osteoclast cells. Resorption is followed by the appearance of osteoblast cells which form bone slowly and act to replace the resorbed tissue. Factors which control bone turnover mediated by osteoclasts and osteoblasts include systemic factors (e.g. hormones, lymphokines, growth factors, vitamins) and local factors (e.g. cytokines, adhesion molecules, lymphokines, growth factors, cytokine inhibitors). These factors, as well as others, tightly control bone turnover and their inactivation may lead to defects in bone formation and turnover.
There are a number of bone disorders associated with defects in the bone turnover cycle. These include osteoporosis, osteoplasia, bone mass loss (osteopenia), Paget's disease, etc. In addition, defects in the bone turnover/repair system can also lead to complications in clinical orthopaedics, for example, fibrous non-union following bone fracture, implant interface failures and large allograft failures. Massive bony defects often occur following trauma involving bone injuries, particularly where the injury is associated with a sudden impact, such as those occurring in motor vehicle and sports accidents. A segmental defect fracture generally ends up in a non-union if it is not treated by extended and complicated surgical procedures.
Conventional bone grafting is currently considered to be the method of choice for the treatment of segmental defect fractures, although the procedure is often unsuccessful. See Albertson et al., Clin. Orthop. 269:113–119 (1991); Zaslav and Meinhard, Clin. Orthop. Rel. Res. 233:234–242 (1988). In addition, bone grafting is often associated with a number of complications, including infections, paresthesias and pain at the grafting site. See Bestrom et al., Clin. Orthop. Rel. Res. 327:272–282 (1996).
Alternative techniques, such as free vascularized fibular grafting, or the use of external fixator techniques have been used to improve the surgical success rate. See Minami et al, J. Recontr. Microsurg. 8:75–82 (1992). A vascularized fibular graft may be superior to a conventional bone graft, but it is technically difficult and occasionally impossible to accomplish. Unsatisfactory results after surgical treatment of posttraumatic segmental bone defects are described in up to 30% of cases. See Moroni et al, Injury 28:497–504 (1997); Südkamp et al, Akt Traumatol. 23:59–67 (1993).
Segmental defects after tumor surgery are even more challenging to surgeons. The area of bone resection is generally large, all osteoconductive and osteoinductive material has been completely removed, and often the only reasonable therapy is an amputation of an extremity because of the complete lack of bony substance.
More commonly, general bone fractures are treated by casting, allowing natural mechanisms to effect wound repair. The treatment of segmental defects and less traumatic bone fractures would be benefitted by new techniques and therapies designed to stimulate the bone regeneration processes and strengthen the fracture repair process.
The patient population suffering from diseases involving loss of bone mass, such as osteoporosis, would also benefit from new techniques and therapies designed to stimulate bone regeneration processes. Osteoporosis is segregated into type Is and type II. Type Is osteoporosis occurs predominantly in middle aged women and is associated with estrogen loss at menopause, while type II osteoporosis is associated with a general advancement of age in both women and men.
An estimated 20–25 million people are at an increased risk of bone fractures due to the loss of bone mass that occurs in osteoporosis. Currently, the major focus for the treatment of osteoporosis is fracture prevention rather than fracture repair. Fractures in the elderly often do not repair quickly and are responsible for morbidity. Therefore, it would be useful to have a treatment for subjects suffering from osteoporosis which focuses on the repair of fractures. In addition, sites of low bone mass in a subject could also be treated prior to a fracture occurring with bone regeneration therapy.
Different osteoinductive and osteoconductive methods are currently under investigation for developing adequate alternatives to bone grafting. One attractive approach makes use of powerful osteogenic properties of certain growth factors. Members of the transforming growth factor β family of growth factors, including the bone morphogenic proteins (such as BMP-2, BMP-4 and BMP-7), have diverse effects on the growth and differentiation of mesenchymal cells, as well as on their ability to synthesize matrix. See Gerhart, et al., Clin. Ortpo. Rel. Res. 293:3170326 (1993); Ripamonti et al., J. Bone Min. Res. 12:1584–1595 (1997); Yasko et al., J. Bone Joint Surg. 74-A:659–670 (1992). An interesting feature of some of these growth factors is their osteoinductivity, the competence to induce bone formation. This has been demonstrated in vivo by the ability of purified recombinant osteoinductive proteins to induce bone formation at heterotropic sites and in different bone defect models. See Lieberman et al., J. Orthop. Res. 16:330–339 (1998) (bone marrow cell line infected with an adenovirus expressing recombinant BMP-2 secreted biologically active BMP-2 and effected heterotropic bone formation in quadricep muscles of immune deficient mice when transplanted thereto); Ripamonti et al., J. Bone Min. Res. 12:1584–1595 (1997) (induction of bone formation shown by the administration of purified recombinant TGFβ1); Sampath et al., Proc. Natl. Acad. Sci. USA 84:7109–7113 (1993) (induction of bone formation shown by the administration of recombinant drosophila proteins homologous to TGFβ).
In addition, the hedgehog family of proteins, described in U.S. Pat. No. 5,844,079, incorporated herein by reference, are also involved in bone formation. Members of the hedgehog gene family were initially characterized as patterning factors in embryonic development but have recently been shown to regulate skeletal formation in vertebrates. Sonic hedgehog, a member of the hedgehog family, regulates the localized production of bone morphogenic proteins and related molecules which initiate chondrocyte and osteoblast-specific differentiation. See Crit. Rev. Oral Bio. Med. 10:40–57 (1999). The amino-terminal fragment of Sonic hedgehog has the ability to induce ectopic cartilage and bone formation in vivo. In addition, ectopic expression of Indian hedgehog induces expression of the parathyroid hormone-related peptide, which together regulate the rate of chondrocyte maturation. Both Indian hedgehog and Sonic hedgehog stimulate osteoblast differentiation. In conclusion, members of the hedgehog family of proteins are osteoinductive proteins which are significantly involved in skeletal formation through multiple actions on chondrogenic mesenchymal cells, chondrocytes, and osteogenic cells. See Crit. Rev. Oral. Biol. Med. 10:477–486 (1999).
The clinical application of such osteoinductive factors may be limited by their short half-lives if administered as purified recombinant proteins to a subject. Gene transfer may be useful in overcoming this problem. The delivery of genes encoding growth factors can provide high, sustained concentrations of these factors locally and for extended periods of time. See Evans and Robbins, J. Bone Joint Surg. 77A:1103–1114 (1995). Moreover, endogenously synthesized proteins, in contrast to exogenously administered recombinant proteins, may have greater physiological effectiveness. See Niyibizi et al., Clin. Orthop. Rel. Res. 355S:148–153 (1998).
Lieberman et al., J. Orthop. Res. 16:330–339 (1998) have used adenoviral vectors comprising a DNA encoding BMP-2 to accelerate healing of a segmental defect. Healing was achieved by cumbersomely delivering the adenoviral vector ex vivo to a murine stromal cell line which was then introduced in vivo by xenografting which had to be performed in an immunocompromised animal. Clinical application of such an ex vivo approach is burdened with complications. Furthermore, the efficacy of such an approach has yet to be demonstrated in an immunocompetant animal using primary cell cultures.
Both U.S. Pat. No. 5,763,416 of Bonadio et al. (the “'416” patent) and U.S. Pat. No. 5,942,496 of Bonadio et al. (the “'496” patent) disclose methods for transferring nucleic acids encoding osteoinductive agents into bone cells in situ and for stimulating bone progenitor cells for the treatment of bone-related diseases and defects. The '416 and '496 patents are limited to a method for transferring a nucleic acid encoding an osteoinductive agent wherein the nucleic acid is part of a composition comprising a structural bone-compatible matrix. Appropriate matrices of the '416 and '496 patents are described as being able to both deliver the gene composition (nucleic acid) and also provide a surface for new bone growth, i.e., the matrix should act as an in situ scaffolding through which progenitor cells may migrate.
Therefore, an improved method for the delivery of a DNA encoding an osteoinductive agent to the site of bone disease or defect which does not require ex vivo transfection/infection followed by xenografting or in vivo co-administration of a bone-compatible matrix is desired.